Development and application of diagnostic NASBA- ELISA for invasive aspergillosis

Abstract:

Objective：To develop a nucleic acid sequence-based amplification coupled ELISA method for detection of invasive as－pergillosis（IA）. Methods：In this experiment，the nucleic acid sequence-based amplification was combined to the high sensitive sys－tem of digoxingenin. The specific 18sRNA of Aspergillus was amplified by the isothermal digoxigenin（DIG）-labeling NASBA process，then the DIG-labeled NASBA amplicons were immobilized on streptavidin-coated microtiter plate by hybridized with a specific bi－otinylated DNA probe. The hybrids were colorimetrically detected by the addition of an anti-DIG antibodies linked to AIP and sub－strate（disodium 4-nitrophenyl phosphate）. The specificity，sensibility and the clinical diagnostic efficiency of this method were eval－uated by detecting the total RNA extracted from the ten-fold serially titrated Aspergillus spores，the RNA extracted from the compar－isons and 82 blood samples（32 positive cases，50 negative cases）. Results：The total RNA extracted from the ten-fold serially titrated Aspergillus spores was detected by NASBA-ELISA. The optical density value was linearly correlated with the logarithm of the spore amount（y=0.278x+0.119，R2=0.887），and this method could detect as little as 1 CFU aspergillums spore. The RNA extracted from the comparisons（E.coli，S.aureus，P.Aeruginosa，C.albicans，C.neoformans） and Aspergillus were detected by NASBA-ELISA. The results showed that NASBA-ELISA was highly specific for Aspergillus detection and did not cross-react with the non-target fungi or bacte－ria. The performance of NASBA-ELISA system was evaluated by detecting blood samples of 82 patients at high risk for IA. The sensitivity and specificity were 80.6% and 80.0%. The NASBA-ELISA A values were assessed by ROC analysis，area under the curve=0.755（95%CI=0.645 to 0.865）. Conclusion：The NASBA-ELISA method can detect Aspergillus sensitively and specifi－cally，so it provides a new way for the molecular epidemiologic investigation and early diagnosis of IA.